Efflux has become increasingly recognized as a mechanism of resistance to antibiotics and other drugs since the investigators' discovery of the tetracycline efflux protein Tet some 20 years ago. Today, a number of Tet efflux proteins (related by primary sequence) have been described which have two halves, each six alpha-helical transmembrane domains, separated by a cytoplasmic loop and specify an exchange of a tetracycline/cation complex for a proton. Active efflux is one of two major mechanisms of tetracycline resistance which have curtailed the clinical effectiveness of this important family of antibiotics. Studies in the Principal Investigator's laboratory focus on the biochemical and molecular basis of efflux by the 43kD class B Tet protein specified by Tn10. The protein has been purified in detergent as a polyhistidine fusion. Recently, it has been successfully crystallized for 2D analysis. This proposal seeks to determine the tertiary structure of the TetA protein through crystallization methods, combining 2D and 3D efforts, by cysteine cross-linking of site-directed cysteine mutant residues, and by genetic analysis of suppressor mutations. In a second aim, they shall pursue findings that implicate the interdomain loop in the function of the Tet protein, using mutagenesis of the loop and cysteine cross-linking studies to examine how the loop interacts with other domains of the Tet protein. A third aim will continue their work on radiolabeled photoaffinity tetracycline analogs to identify the substrate-binding region in the Tet protein. This approach will be complemented by studies that use iron as the cation for tetracycline efflux and the Fenton reaction to produce peptide bond cleavage at substrate binding sites in the protein. These studies pursue further the molecular basis for antiport by the drug efflux protein Tet. Advances in understanding of the Tet protein will enhance our knowledge of other, similar proteins in the major facilitator family of transport proteins which should have broad impact in the area of drug treatment of infectious disease and cancer.